This invention relates to protein structure, and more particularly to proteins involved in amyloidogenic disorders.
Alzheimer""s disease and spongiform encephalopathies are examples of conditions in which specific proteins transform from their native states into bundles of ordered fibrils termed amyloid. Protein concentration, point mutations, and solvent composition influence fibril formation, but a structural feature unique to amyloid-forming protein has not been detected.
Amyloid fibrils can be formed from different proteins. They are associated with neurodegenerative disorders such as Alzheimer""s disease and the prion diseases (e.g., Creutzfeld-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephelopathy), as well as other organ-specific and systemic amyloidoses (The Merck Manual, 16th ed., Merck Research Laboratories, Rahway, N.J., 1992, pp. 1052-1053; Kelly, 1996, Curr. Op. Struct. Biol. 6:11-17). The proteins involved in forming amyloid are constitutively present in a soluble state, but form insoluble aggregates under certain conditions (Chiti et al., 1999, Proc. Natl. Acad. Sci. USA 96:3590-3594). There are no obvious common properties in amino acid sequence, three dimensional structure, or function among the approximately 20 proteins that are known to be specifically associated with amyloid diseases (Sipe, 1992, Annu. Rev. Biochem. 61:947-975). In spite of the differences in native structures, the amyloid fibrils are similar, irrespective of the protein from which they originate (Dobson, 1999, TIBS 24:329-332). Amyloid fibrils are built up from a cross-xcex2-scaffold, with xcex2-strands perpendicular and xcex2-sheets parallel to the fiber axis.
Amyloid diseases mostly occur without known precipitating factors (Lansbury, 1999, Proc. Natl. Acad. Sci. USA 96:3342-3344). Destabilizing point mutations can cause fibril formation of an otherwise stable protein, e.g., in lysozyme (Booth et al., 1997, Nature 385:787-793), but point mutations related to inherited forms of human prion diseases do not induce PrPSc (the disease-associated form of a prion) in vitro and are not generally destabilizing (Liemann et al., 1999, Biochemistry 38:3258-3267). The so-called Axcex2 (e.g., 1-42 residue) peptide associated with Alzheimer""s disease is highly fibrillogenic, while peptides lacking residues 14-23 are not (Tjernberg et al., 1999, J. Biol. Chem. 274:12619-12625).
The invention relates to the discovery that a polypeptide containing an amino acid sequence that is predicted to be able to undergo a conversion from xcex1-helix to xcex2-strand can form fibrils. An amino acid sequence that is present as a helix in a polypeptide but is predicted to form a xcex2-strand structure is herein termed a discordant helix. Compounds that stabilize the xcex1-helical form of a discordant helix are useful for treating disorders in which xcex2-strand structures form fibrils. Such disorders include amyloidoses such as prion diseases and Alzheimer""s disease. The invention includes methods of identifying discordant helixes, methods of identifying compounds that can stabilize the xcex1-helical form of a discordant helix, and compounds identified by these methods. The invention also includes methods of treating disorders in which xcex2-strand structures are a part of the pathology of the disorder, e.g., amyloidoses. Such disorders include Alzheimer""s disease and prion-associated diseases (e.g., scrapie, bovine spongiform encephalopathy, and Creutzfield-Jacob disease).
The invention features a method of identifying a compound that stabilizes an xcex1-helical conformation of a discordant helix in a polypeptide. The method includes the steps of providing a test sample containing a polypeptide that contains a discordant helix in the form of an xcex1-helix, contacting the test sample with a test compound, and determining the rate of decrease in the amount of xcex1-helix in the test sample, such that a lower rate of decrease in the presence of the test compound than in the absence of the test compound is an indication that the test compound stabilizes the xcex1-helical conformation of the discordant helix in the polypeptide. The invention also includes compounds identified using this method. Test compounds that can be used according to the method include peptides, e.g., tripeptides such as dipolar tripeptides. In those embodiments where the polypeptide containing a discordant helix includes all or part of an Axcex2 peptide, the polypeptide can include at least residues 14-23 or 16-23 of the Axcex2 peptide.
The invention also feature a method of identifying a compound that can stabilize an xcex1-helical conformation of a discordant helix-containing polypeptide in which the method includes providing a test sample comprising a polypeptide that contains a discordant helix in the form of an xcex1-helix, contacting the test sample with a test compound for a specified amount of time, and determining the amount of xcex1-helix present in the test sample such that a larger amount of xcex1-helix in the presence of the test compound than in the absence of the compound indicates that the test compound stabilizes the xcex1-helical conformation of the discordant helix in the polypeptide. The invention also includes compounds identified using this method. Test compounds that can be used according to the method include peptides, e.g., tripeptides such as dipolar tripeptides. In those embodiments where the polypeptide containing a discordant helix includes all or part of an Axcex2 peptide, the polypeptide can include residues 14-23 or 16-23 of the Axcex2 peptide.
The invention also features a method of identifying whether a protein is susceptible to forming amyloid which includes analyzing the amino acid sequence of the protein to determine whether the protein contains a predicted discordant helix, such that the presence of predicted discordant helix is an indication that the protein is susceptible to forming amyloid. The discordant helix can be at least six amino acids in length.
The invention includes a method of decreasing the rate of formation of xcex2-strand structures between at least two discordant helix-containing polypeptides, in which the method includes contacting the discordant helix-containing polypeptides with a compound that stabilizes an xcex1-helical form of the discordant helix. Tripeptides such as the dipolar tripeptides described herein can be used to stabilize the discordant helix.
The invention also features a method of treating an individual having or at risk for an amyloidosis. The method includes administering to the individual a therapeutically effective amount of a compound that stabilizes an xcex1-helical form of a discordant helix-containing polypeptide that forms amyloid. The amyloidosis can be, for example, a prion disease or Alzheimer""s disease. Tripeptides, e.g., dipolar tripeptides including those described herein, can be used to stabilize the discordant helix.
A xe2x80x9cdiscordant helixxe2x80x9d is an amino acid sequence that is predicted to be able to form an xcex1-helix and is also predicted to be able to form a xcex2-strand. A discordant helix can be identified using structure analysis programs that predict secondary structure of polypeptides, specifically by analyzing an amino acid sequence for predicted xcex1-helix and also analyzing the amino acid sequence for predicted xcex1-strand. A sequence that is predicted to form xcex1-helix and xcex2-strand is a discordant helix. A discordant helix amino acid sequence can be an isolated peptide, or form part of a polypeptide. A discordant helix can be naturally occurring in a wild type or mutant polypeptide. A discordant helix can also be in a synthetic amino acid sequence. In general, the discordant helix amino acid sequence is at least about 6 amino acids in length. Such sequences can be longer, e.g., 7, 8, 9, 10, 11, 12, 14, 16, 18, 22, 24, or 26 amino acids in length.
A xe2x80x9cpolypeptidexe2x80x9d means a chain of amino acids regardless of length or post-translational modifications.
A xe2x80x9cnon-amyloidogenic formxe2x80x9d of a polypeptide containing a predicted discordant helix is the form of the protein in which xcex1-helix is the predominant conformation of the discordant amino acid sequence. Compounds that promote the xcex1-helix conformation of a discordant helix are useful for preventing the formation of amyloid.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials suitable for practicing the invention are described below, method and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description, and from the claims.